Jump to Content

Living Neural Networks

We are interested in learning, memory, and synaptic plasticity in living neural systems, either in dissociated cell cultures, organotypic cultures (e.g., hippocampal slices), or living animals. Professor Pine and his collaborators have developed a number of new technologies for studying neural systems. Examples include an x-ray microscope for living cells, multi-electrode cell culture chambers, fiber-optic photodiode arrays and high-speed CCD cameras for imaging of neural activity, and silicon probes for long-term interfacing of neural tissue with external electronics. The projects below describe our continuing efforts to advance the study of real neural networks. Here is a more detailed (but dated) synopsis of the work, from the Division of Biology's brochure.

Prof. Jerome Pine

Professor of Physics in the Division of Physics, Mathematics, and Astronomy, currently residing in the Division of Biology.

Prof. Pine is also the Co-president of the Caltech Pre-college Science Initiative, along with Prof. Jim Bower.

Steve M. Potter, PhD

Senior Research Fellow in the Division of Biology

I am a member of the Research Faculty working jointly in the Pine lab and the lab of Prof. Scott Fraser. I am pursuing a number of projects concerning the ways in which neuronal morphology and synaptic plasticity interact, and how they lead to learning and memory. I am very interested in studying how ensembles of neurons represent and process information, using multi-unit approaches such as

In 1994, I put together a 2-Photon laser-scanning microscope that we have used to image a wide variety of living specimens, and to produce time-lapse movies of neurons growing and changing in brain slices, embryos, and dissociated neural cultures.

I previously worked on the Neural Probe project as part of the NIH Neural Prosthesis Program.

Since April 1999, I have been the Principal Investigator on an RO1 grant from the NIH, and have a semi-independent research group within the Pine and Fraser labs.

Visiting Post-doc

Chris Lindensmith

Grad Student in Physics

Daniel Wagenaar

Daniel Wagenaar (wagenaar@caltech.edu)

Formerly in the lab:

  • Michael P. Maher, PhD

    Neurochip and dissociated hippocampal cultures.

    More about Mike Maher and the Neurochip project. Mike is now at Aurora Biosciences in San Diego, California.

  • Hannah Dvorak-Carbone

    Neurochip and dissociated superior cervical ganglion cultures. Hannah has recently finished her PhD in the Schuman Lab.

  • Wade G. Regehr, PhD

    Reid Phto

  • Chi-Bin Chien, PhD

    The goal of Chi-Bin's thesis work was to study long-term synaptic plasticity in small networks of cultured neurons. He developed methods for making microcultures of 3 to 10 rat sympathetic neurons grown on small laminin islands. These neurons become cholinergic in culture and, confined to these islands, formed many synapses with each other. To record noninvasively from many cells simultaneously, he used optical recording with voltage-sensitive fluorescent dyes, using a custom-built a 256-photodiode optical detector. The extremely low noise of the preamplifiers made it possible to record fluorescence signals limited only by photon-counting statistics, and thus detect 5-10 mV changes in membrane potential (Chien and Pine, 1991a). These were the most sensitive optical recordings that had then been made. Accurate measurement of synaptic strengths was however precluded by the complex anatomy of the cultures: signals from synaptic potentials were often obscured by signals from action potentials in axon potentials (Chien and Pine, 1991b).

    References:

    Chien, C.-B., and Pine, J. (1991a) An apparatus for recording synaptic potentials from neuronal cultures using voltage-sensitive fluorescent dyes. J. Neurosci. Meth. 38:93-105.

    Chien, C.-B., and Pine, J. (1991b) Voltage-sensitive dye recording of action potentials and synaptic potentials from sympathetic microcultures. Biophys. J., 60:697-711.

    Present address:

    Dept. Neurobiology & Anatomy
    409 Wintrobe
    Univ Utah Medical Center
    50 North Medical Drive
    Salt Lake City, UT 84132

    (801)585-1701 phone
    (801)581-4233 fax

    chi-bin.chien@hsc.utah.edu

    http://www.neuroscience.med.utah.edu/Faculty/Chien.html

  • Tina Garyantes

    My PhD dissertation work at Caltech consisted of looking at the effect of electrical stimulation, via a cell attached patch, on cell growth and intracellular calcium concentrations in SCGs. We found that neither electrical stimulation at up to 10 Hz nor the concurrent increase in calcium concentration in the growth cone affect neurite outgrowth. This finding was counter to the prevailing wisdom, determined from Helisoma neurons, at the time. I also worked on the development of the wells. In particular, I developed some interesting fabrication methods centered on providing polyimide covers to the wells, I was able to both stimulate and record from SCGs in the wells but not both simultaneously.

    Reference

    Garyantes, T. K. and Regehr, W. G. (1992) Electrical activity increases growth cone calcium but fails to inhibit neurite outgrowth from rat sympathetic neurons. J Neurosci 12: 96-103.

    Outside of the Pine lab, I was group leader of process development at Cryopharm during graduate school. Since then I worked for Ciba Corning/ Chiron Diagnositics for 3 years where I designed instruments for genomic fine structure analysis as the group leader of systems development. Currently, I am at Merck in combinatorial chemistry where I develop novel systems for miniaturized high throughput screening.

How to eavesdrop on cultured neurons:

The Neurochip

In collaboration with Prof. Yu-Chong Tai of Caltech's Electrical Engineering department, we have fabricated silicon devices for maintaining long-term (weeks or months) connections between cultured neurons and external stimulation and recording electronics. Neurons dissocated from embryonic rat brain tissue are placed wells in the silicon substrate, in contact with gold electrodes at the base of the wells. Once a neuron grows and sends out processes, it is trapped in the well by the grillwork that covers the well, as shown below:

Rat superior cervical ganglion neuron growing from a silicon well on the Neurochip. A process from a neuron in a nearby well is visible to the lower left of the well. Click on the image to download a time-lapse movie of the cell in action (280K flattened QuickTime).

Each Neurochip has 16 wells, in a 4 x 4 array, designed to hold a single neuron each. By holding the neuron in close proximity to the extracellular electrode, we avoid problems encountered with flat-dish electrode arrays, due to neurons migrating away from the electrodes as they grow and wander. Thus, the Neurochip will provide an unprecedented ability to monitor and influence the synaptic activity of a small network of neurons, and to discover the effects of neural activity on synaptic plasticity.

Click here for the latest on the Neurochip project.

Connecting brains to computers:

The Neuroprobe

The masters of microfabrication in Prof. Tai's lab have also produced silicon probes designed to hold 15 neurons in a line of wells, like the wells of the Neurochip. The Neuroprobe, part of the Neural Prosthesis Program of the National Institute of Neurological Disorders and Stroke (National Institutes of Health), is designed to be permanently implanted into neural tissue. When the neurons in the wells send out processes and make synaptic contacts with the host tissue, we will have a specific, long-term connection with the tissue for stimulation and recording studies. We collaborate with Prof. Gyorgy Buzsaki at Rutgers University, who is testing the probes in living rats. In our lab, Steve Potter is using cultured hippocampal slices from neonate rats as the host tissue, so that outgrowth from the probe and synaptic integration with the slice can be monitored non-destructively, over time.

Probes

To help us choose healthy neurons to be put into the wells of the Neurochip and Neuroprobe, we have made time-lapse movies of hippocampal neurons establishing themselves on gridded petri-dishes coated with polylysine and laminin.

Click on the image to download such a time-lapse movie, spanning the first few hours after plating the cells (Caution: 3Mb flattened QuickTime. 640x480 greyscale).

Links of Interest:

November 1999 Steve Potter (spotter@gg.caltech.edu)